Colony shape as a genetic trait in the pattern-forming Bacillus mycoides

Strains

We started by isolating B. mycoides strains from the wild. A soil sample from the Department garden was diluted into sterile water and plated on agar plates. Among colonies of several microorganisms, filamentous colonies as described for B. mycoides[12, 13] were found. Most displayed projections curving to the left hand (SIN), as seen from the bottom of agar plates, and fewer to the right hand (DX). Since colonies are in general observed from the bottom of the agar plate, due to vapor fuzziness under the lid, the names (DX-dextral and SIN-sinistral) were given to colonies seen in this way. On the agar surface the opposite is visible, that is DX filaments are curving counter-clockwise and SIN clockwise. Isolated single colonies were inoculated in liquid and solid media and two strains were selected for study. Colonies of the two forms are shown in Fig. 1.

Positioning of the Petri dish at different angles towards the bench top did not change colony geometry of the strains. Experiments were conducted to see if curvature direction could be modified by a reduced gravity (microgravity). For this purpose colonies were grown on agar Petri dishes subjected to conditions of simulated microgravity, tied to the revolving plane of a three-dimensional clinostat. No modification of the colony patterns was observed (unpublished results).

Since we were dealing with environmental isolates, we used the RAPD (Random Amplified Polymorphic DNA) test to confirm by a molecular marker that strains belonged to B. cereus group. This group is composed by B. cereus, B. thuringiensis, B. anthracis and B. mycoides[14]. Total DNA was amplified from our isolates and from B. mycoides NRRL NRS 273^T, B. pseudomycoides NRRL B-617^T, B. thuringiensis BGSC 4D1, B. cereus Pasteur 6452 and B. subtilis BD366 (as a negative control), using the same primers. Primers had been designed as diagnostic for B. cereus group and negative for a panel of Bacilli[15]. We found the expected specific 749 bp band in all strains, except for B. subtilis (Fig. 2).

Biochemical features of the new isolates were analyzed, along with B. mycoides Type strain, with the API50CH test (bioMerieux) that monitors 50 different enzymatic reactions using sugars as substrates. SIN and DX enzymatic activities were in the range indicated by the test for B. mycoides. A few minor differences of color intensity between SIN and DX were in the variability range reported for B. mycoides species (not shown). As in the case of the strains studied by Gause [12] our SIN strain was unable to hydrolyze sucrose.

Several growth conditions were tested to check for colony morphotype stability. Different liquid and solid media (see Materials and Methods) all supported growth of the isolated strains and their specific colony pattern. Media at pH values ranging from 4.0 to 9.0 were tested: up to pH 5.0 the strains are unable to grow, nor do they resume growth when transferred to standard conditions. At pH 5.6 and over they can grow, the preferred range being between 6 and 8.5.

Temperature range for DX and SIN growth was also analyzed. Both divide from 6°C to 37°C, with an optimum at 26–28°C. 38°C exert a bactericidal effect on DX, but not on SIN, which can survive, though not divide, up to 42°C, some cells being able to restart growth at lower temperature. Temperature over 35°C is deleterious for colony shape: both DX and SIN reduce filament length and at 37°C no turn direction is observed.

In liquid media the typical morphology of SIN and DX is an aggregated phenotype: bacilli form small round clumps when well agitated and a single big clump when kept still. The surrounding liquid is clear and the aggregates can be disrupted only by very vigorous vortexing (Fig. 3).

The SIN and DX strains were analyzed for the presence of natural plasmids: two were found in SIN, 9.2 and 3.5 kb in length, while DX harboured 4 plasmids, 13.8, 10.5, 10.0, and 3.3 kb long. The smallest plasmids of the two strains were cloned and sequenced (EMBL acc.#AJ243967 and AJ272266). They belong to rolling circle replicating plasmids commonly found in Gram-positive bacilli. Regions homologous to single-strand and double-strand origins of replication (ss-ori and ds-ori) of B. cereus group plasmids are present. Open reading frames showing similarity to Rep and Mob proteins of many bacteria are present in both plasmids in the same position and orientation [16].

A derivative of SIN called SIN96, which lost the larger plasmid but kept wild type morphotype, was the starting strain for the search of morphotype mutants.

Morphotypes of the SIN strain

The first spontaneous mutant was a SIN strain that lost turn direction of the filaments on agar plates and aggregation in liquid culture (Fig. 3). Similar non-aggregated morphotypes appeared at enhanced frequency by culturing in liquid with Novobiocin, a drug used to cure strains of the endogenous plasmids [17]. Morphotypes were found to fall into two main types: colonies with no turn direction of macroscopic protrusions, but still rhizoidal (that we define "cotton-like" due to their fluffy appearance), and colonies with a round and compact shape, similar to those of B. cereus. One example is SINett, followed by light and electron microscopy during colony formation. Other mutants, occurring only once during screening, were: CIC, which neither lost aggregation in liquid culture, nor turn direction of the filaments, but had shorter and thicker trunks and CAD, no more rhizoidal, with reduced adherence to agar (Fig. 4). SIN96, with wild type morphotype, is in the center of Fig. 4 and some morphotype mutants at the periphery. All were inoculated at the same time with the same cell numbers. The reduced degree of space colonization of the mutants compared to wild type is apparent. Closer inspection showed that apparently similar mutants differ one from the other, mostly among cotton-like colonies, as shown in Fig. 5.

Mutant colonies were checked for ribosomal DNA EcoRI restriction pattern (ribotyping) to ascertain strain lineage and lack of gross genomic rearrangements. Location of ribosomal clusters inside the genome gives a reproducible pattern of restriction bands which is useful to trace single colonies as belonging to the same strain [2]. EcoRI digested total DNA was hybridized to a synthetic oligonucleotide chosen in the very conserved part of 16S rDNA of group I Bacilli [18]. In Fig. 6, ribotyping of several Bacillus strains shows that they differ for many bands, while all of the mutant colonies derived from SIN96 maintain the parental pattern.

Formation of colonies on agar

With the help of an optic microscope we could follow colony formation starting from one or a few cells seeded on agar plates. Figures 7, 8 and 9 show a series of pictures which depict colony formation of wild type SIN and DX strains and of the SIN mutant with round compact colonies, SINett. We examined large numbers of colonies during growth and the examples in the panels reflect the typical pattern of each strain.

Wild type SIN is shown in Fig. 7. A lag period of 1–4 hours usually follows seeding of single colony forming units: here, a filament containing a few cells is visible after about two hours (Fig. 7/2). In this colony a ramification, due to a new cell growing from a splitting of the filament, and an angle appeared (shown by arrows in Fig. 7/2). These were the reference points that, by remaining at the same mutual distance, made clear that growth was due to division of the edge cells only. In fact, the number of cells remains the same inside the central structure (see insets of Fig. 7/4 and 7/5). After 5 hours (Fig. 7/6) a curvature appears at both edges, typically turning clockwise. At later times new filaments running parallel to the previous ones arise at breakage points between two adjacent cells. Others start at an angle with the founder filament and expand towards empty spaces. After 10 hours many filaments run in parallel with the main trunk and new curly trunks are present on the plate.

Fig. 8 shows a DX colony. As for SIN, the filament grows from the edges, that both curve counterclockwise. The first ramification occurs at later times (Fig. 8/5). Insets show one mode of rupture formation: a filament following the upper ramification first bends and then breaks forming two growing points that eventually duplicate the founder filament and the ramification. In Fig. 8/6 additional ramifications appear and the circle is formed with more than one filament (see insets). In Fig. 8/7 many filaments follow the road opened by the founder filament and many lateral filaments emerge. After 16 hours (Fig. 8/8) the initial shape of the founder filament is still recognizable, together with new big trunks with the strain specific curvature. In Fig. 8/9, at 72 hours, the plate surface is completely covered.

Colony formation of these two wild type strains follows the rule of leaving the initial location on the agar surface towards a centrifugal direction, mostly by division of the outer cells.

The mutant SINett behaves differently (Fig. 9). At first cells divide in a linear array like the wild type, but, after a short time, cells internal to the string divide creating tensions that make cell edges detach one from the other and form ruptures that become new growing edges. The process is repeated again and again, to fill all of the space close to the founder filament until it is completely covered. No growth occurs at the outer cells of the first filament which remain at about the same distance. The cells that remain outside the growing mass are often subjected to autolysis, leaving the colony borders without projections (Fig. 10).

Scanning electron microscopy

B. mycoides colonies on agar were observed also at the scanning electron microscope. SIN96, with wild type morphotype, is shown in Fig. 11. Migration of many filaments out of the colony mass is apparent at low magnification in Fig. 11a. Details of the same region are shown in b, c, d, e, f. Cells at the growing edge move in two different ways. Some cross one over the other following independent routes (11 b) while others converge along the way (11c). Filaments can join and then separate, resembling motorway junctions (Fig. 11d). No twisting of filaments one around the other, nor of cells around their axis is visible, even at high magnification (Fig. 11e and 11f).

Morphology of the mutant SINett on agar is shown in Fig. 12. The shape of an entire colony, made by filaments forming parallel wavy bundles around the colony center, is visible in Fig. 12a. Details of the same colony are in panels 12b, 12c, 12d: filaments contain cells completely separated one from the other, much more frequently than in wild type colonies. Some minicells are present. The basic structure of the mutant is a filamentous organization as in the wild type, even though these round compact colonies appear at the macroscopic level similar to those of non-filamentous bacteria.

B. mycoides dcw gene cluster

The genes coding for the proteins assembling at the division site of bacillar cells are mostly clustered, together with those specifying enzymes for the synthesis of peptidoglycan. The cluster dcw (d ivision c ell w all) is well conserved among bacilli with respect to gene order [19]. Very little is known about B. mycoides at the genomic level: knowledge of the genes involved in fundamental steps of cell division appears as a necessary base for studying the processes of colony assembly. We therefore started to characterize the dcw cluster in DX and SIN strains.

Due to high conservation of the main protein of the cluster, FtsZ, in bacteria and even in eukaryotic chloroplasts [20] we could design forward and reverse degenerate oligonucleotides inside two regions of the protein with maintained amino acid sequences. Amplification by PCR of total DNA of DX and SIN strains gave a DNA fragment whose sequence had high homology to the corresponding region of B. anthracis ftsZ gene (sequences provided by TIGR, The Institute for Genomic Research). Homology was such that we could subsequently design oligonucleotides based on B. anthracis genome for chromosome walking upstream and downstream of the ftsZ sequence first determined.

Fig. 13 shows comparison of DX and SIN amino acid sequences along four genes, ftsQ, ftsA, ftsZ and murC. DNA spacer sequences (not shown) between genes are less conserved than coding sequences. In particular the spacer that separates ftsZ from murC is much longer in the SIN strain compared to DX and to B. anthracis. The same spacer region was analyzed in B. mycoides Type ATCC 6462 strain and in B. pseudomycoides Type B617 strain [21]. The ATCC 6462 Type strain is 100% identical even along this spacer to DX, once more confirming classification of the strain. The more divergent was found to be B. pseudomycoides, whose colonies are very similar to those of B. mycoides DX, with the same direction of filament curvature. The lower similarity at the gene sequence level confirms a greater genetic distance of this species from the other members of B. cereus group [14].

Transformation

For every genetic approach the possibility to transform strains with DNA is essential. Many protocols of protoplast transformation and of electroporation were tested on our environmental strains with no success. With a few modifications of the Macaluso protocol [22] two strains, SINV6 and Ett, were transformed with the recombinant plasmid SIN-HPS9 (not shown). It is made by the union of the shuttle vector HPS9, carrying two antibiotic resistances [23] with the smallest 3.4 kb cryptic plasmid of the SIN strain, pBMY1 [16]. Efficiency however was very low and needs to be further improved.

Isolation of B. mycoides strains

A soil sample was collected and suspended in sterile water. Aliquots were plated on agar plates. Selected colonies with filamentous growth in the clockwise or counterclockwise direction, SIN and DX, were passed for several generations in liquid and solid media. Routine cultures were on Trypton soya or LB [24], liquid or agar (15 g/L agar). Bacillus cereus selective medium, Brain heart infusion, Blood agar, Muller Hinton were also occasionally utilized: these media were from OXOID (Unipath Hampshire)

RAPD screening

The environmental strains were tested by PCR amplification of total DNA with primers producing a 749 bp fragment diagnostic for the Bacillus cereus group [15]. 10 ng total DNA was amplified for 35 cycles (1 min at 94°C, 1 min at 45°C, 2 min at 72°C) with primers 5'-ACTGGCTAATTATGTAATG-3' and 5'-ATAATTATCCATTGATTTCG-3' in 25 μl final volume containing 2.5 μl 10X buffer (Perkin-Elmer), 200 μM each dNTP, 1.5 mM MgCl2, 1.0 μM primers and 1.5 U. AmpliTaq polymerase (Perkin-Elmer), covered with 50 μl mineral oil. Samples were analyzed on 1% agarose gels in TBE buffer [24].

API 50 CH strip test

Liquid cultures of SIN and DX were analyzed with the bioMerieux API 50 CH test strips in parallel with liquid cultures of B. mycoides and B. pseudomycoides[21] (strains NRRL NRS-273 and NRRL B-617^T respectively, kindly provided by L. K. Nakamura, Dept. of Agriculture, Peoria IL).

DNA isolation and ribotyping

Bacterial pellets were lysed in 0.1 M EDTA, 0.1 M Tris-HCl pH 9.0, 1% SDS at 65°C for 1 hour. The viscous solution was made 1 M K-acetate, incubated 30 min in ice and centrifuged. Supernatant was precipitated with 0.7 volumes isopropanol and washed twice with 80% ethanol. DNA was digested with EcoRI, electrophoresed on 0.8 % agarose in TBE and blotted to Amersham Hybond N⁺. The filter was hybridized with a 20 mer oligonucleotide with the sequence 5' GAGTTTGATCCTGGCTCAGG 3', from the conserved 16S region of the group I bacilli (B. cereus, B. thuringiensis, B. anthracis, B. mycoides), 5'-end labelled with ³²P-γATP and polynucleotide kinase.

Preparation of B. mycoides plasmids

Cultures were grown to the mid-log phase (A _{600 nm} = 0.4) in LB at 30°C and cells pelleted, washed with isotonic buffer and incubated for 1 hour at 37°C with light shaking in sol. A (20% sucrose, 10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM EDTA, 10 mg/ml lysozyme, 100 μg/ml DNase free RNase). Cells were treated at room temperature with 0.2 N NaOH, 1% SDS for about 10 min. The solubilized cell content was neutralized with 5 M K-acetate pH 4.8 and after 30 min at 0°C the supernatant cleared by centrifugation and nucleic acids precipitated with 0.7 vol. isopropanol. The pellet was washed with 80% ethanol, resuspended in TE and run on a 0.8% agarose gel with supercoiled plasmids (BRL) as reference M.W. markers.

Curing techniques

Two different methods were employed for inducing loss of the plasmids of the SIN strain. High temperature: cells were plated in Petri dishes with agar (0.8 g/l) in LB and incubated 8–18 hr at 37°C. Treatment with Novobiocin [17]: cells were treated in liquid LB at 30°C for 48–72 hr with 0.2 μg/ml of Novobiocin (Sigma), with daily changes of the antibiotic containing medium.

Light microscopy

An inverted Zeiss Axiovert 25 and a digital camera Nikon Coolpix 990 were used for colony observations.

Scanning electron microscopy

Colonies of SIN and DX, grown for a few hours at room temperature on the surface of 1.5% agar LB plates, were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer as minute square pieces. Samples were rinsed with buffer and postfixed in 1% OsO₄ in Zetterquist buffer for 5 hours. After washing in buffer, they were dehydrated by passage for 30 min each through a graded ethanol series. After critical point drying, samples were coated with gold. Samples from liquid cultures were fixed and postfixed on glass coverslips like the colonies, with shorter dehydration times. Samples were examined in a Hitachi (Tokio, Japan) S-570 scanning electron microscope.

Sequencing

Oligonucleotides for starting FtsZ sequencing in SIN and DX were designed inside two very conserved amino acid tracts of the FtsZ protein [20]. Nucleotide sequences of these regions in B. halodurans, B. subtilis, L. lactis and B. anthracis (from TIGR) were considered for synthesising two degenerated primers: Zdir, 5' AAAGTWATCGGTGTCGGCGGWGGC 3' and Zrev, 5' CRGAAATMCCTTGAACMCCTTGRCG 3'. 200 ng of total DNA from DX and SIN were amplified in 50 μl mix, containing 1.5 mM Mg, 0.2 mM dNTP, 0.8 mM primers, 5 units of Amplitaq (Perkin Elmer). Conditions were: 5 min at 95°C; 30 cycles: 1 min 94°C, 1 min 65°C, 1 min 72°C. PCR products were purified from agarose gels by the Concert™ Rapid Gel Extraction System (Gibco BRL) and sequenced by M-Medical sequencing service. For further sequencing, 20 bp oligonucleotides were synthesized based on DX and SIN sequences and on B. anthracis unfinished whole genome sequence – preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org – and used in pCR reactions as above. When primers did not work on B. mycoides DX and SIN DNA, they were made degenerated in the third base when complementary to a coding B. anthracis sequence.

Transformation

We followed the protocol by Macaluso et al. [22] with the following modifications: cells in the exponential growth phase were used; electroporation cuvettes were 0.2 cm instead of 0.4 cm wide, at 100-Ohm instead of 5-Ohm; growth in non-selective medium after electroporation was extended to 3 hours and growth in selective liquid medium was for 5 days. Recombinant plasmid SIN-HPS9 used for transformation was obtained by inserting NdeI digested pBMy1 [16] into the unique NdeI site of the shuttle vector pHSP9 [23]. The recombinant plasmid was amplified in Bacillus subtilis BD366 (BGSC-1E6). Less than 200 B. mycoides transformants were obtained per microgram DNA.